Int J Hematol (2014) 100:341–344 DOI 10.1007/s12185-014-1661-4
RAPID COMMUNICATION
Induction of immune tolerance to platelet antigen by short-term thrombopoietin treatment in a mouse model of immune thrombocytopenia Tetsuya Nishimoto • Miku Numajiri • Hisataka Nakazaki • Yuka Okazaki • Masataka Kuwana
Received: 26 June 2014 / Revised: 31 August 2014 / Accepted: 1 September 2014 / Published online: 12 September 2014 Ó The Japanese Society of Hematology 2014
Abstract Immune thrombocytopenia (ITP) is an autoimmune disorder caused by IgG anti-platelet autoantibodies. Thrombopoietin (TPO) receptor agonists are highly effective in inducing the recovery of platelet counts in ITP patients. Although these agents are thought to promote platelet production without affecting the autoimmune pathogenesis of the disease, a small subset of ITP patients exhibits sustained platelet recovery after treatment termination. To investigate mechanisms involved in this sustained recovery, we evaluated the effects of short-term TPO treatment using a mouse ITP model generated by Foxp3? T regulatory cell (Treg) depletion. After treatment, platelet recovery was sustained, along with complete suppression of both anti-platelet autoantibody production and T-cell responses to platelet autoantigens. TPO treatment also promoted the peripheral induction of Foxp3? Tregs in conjunction with elevated circulating TGF-b levels. In summary, thrombopoietic agents are capable of inducing immune tolerance to platelet autoantigens, thereby suppressing the autoimmune pathogenesis of ITP. Keywords Immune thrombocytopenia Immune tolerance Platelets T regulatory cells Thrombopoietin
T. Nishimoto M. Numajiri H. Nakazaki Y. Okazaki M. Kuwana Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Y. Okazaki M. Kuwana (&) Department of Allergy and Rheumatology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan e-mail: [email protected]
Introduction Immune thrombocytopenia (ITP) is an autoimmune disorder caused by the production of IgG autoantibodies to platelet membrane glycoproteins, such as GPIIb/IIIa and GPIb [1], which depends on activation of pathogenic autoreactive CD4? T cells [2]. In ITP patients, various immunosuppressive processes, including T regulatory cell (Treg)-mediated immune regulation, are dysfunctional, resulting in autoreactive T-cell activation [3]. We recently demonstrated that Foxp3? Treg-deficient mice spontaneously develop sustained thrombocytopenia associated with IgG anti-GPIb antibody production [4, 5], analogous to the pathophysiology of human ITP. This mouse model is useful for evaluating novel therapeutic strategies for ITP. Thrombopoietin (TPO) regulates thrombopoiesis through the activation of megakaryocytes in the bone marrow, resulting in increased platelet production [6]. Recently, the development of TPO receptor agonists (TPORAs), such as romiplostim and eltrombopag, was a significant breakthrough in ITP treatment [6]. It is thought that TPO-RAs do not affect the autoimmune pathogenesis of ITP, since platelet counts typically drop to pre-treatment levels immediately after treatment termination. However, recent reports showed that platelet recovery was unexpectedly sustained in some patients even after TPO-RA was discontinued [7–9]. The exact prevalence of TPO-RAinduced sustained remission is unclear, but one report described that this occurred in 3 of 31 patients treated with TPO-RAs [9]. Based on this observation, a single course of dexamethasone in combination with eltrombopag has been evaluated for a potential therapeutic strategy that induces long-term remission in newly diagnosed ITP patients [10]. Here, we investigated mechanisms mediating the sustained
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effects of thrombopoietic agents using our mouse ITP model.
Materials and methods Recombinant TPO (rTPO) treatment of Treg-deficient ITP mice Treg-deficient mice were established by transferring CD4?CD25- T cells into syngeneic nude mice, as previously described [4]. Four weeks after transfer, ITP mice, which were confirmed to have thrombocytopenia (platelet count B0.33 9 106/lL), were treated with intravenous administration of mouse rTPO (300 ng/mouse; kindly provided by Kyowa Hakko Kirin, Tokyo, Japan) or vehicle for five consecutive days. Experimental protocols were approved by the Keio University Ethics Committee for Animal Experiments. Evaluation of autoantibody production and autoreactive T-cell responses in splenocytes The production of pathogenic IgG anti-platelet antibodies in splenocyte cultures was assessed using a platelet/IgGbinding assay, as described previously [4]. The T-cell response to GPIba was measured as previously described [2], with some modifications. Briefly, five different overlapping regions encompassing all 734 amino acid residues (aar) of GPIba were expressed and purified as recombinant maltose-binding protein (MBP) fusion proteins [11]. These included a1 (aar 18–250), a2 (aar 242–400), a3 (aar 399–570), a4 (aar 558–674), and a5 (aar 666–734). The splenocytes were cultured in triplicate with the recombinant GPIba fragments or MBP (5 lg/mL) for 7 days. T-cell proliferation was determined by 3H-thymidine incorporation, and the antigen-specific T-cell response was expressed as a stimulation index (SI), calculated as the ratio of the cpm incorporated in the presence of each GPIba fragment to the cpm incorporated with MBP. Phytohemagglutinin (PHA) was used to demonstrate non-specific T-cell responsiveness. Detection of Foxp3? Tregs Splenocytes were fixed, permeabilized, and incubated with fluorescence-conjugated monoclonal antibodies to CD4 (BD Biosciences, San Diego, CA, USA), CD25 (BD Biosciences), and Foxp3 (eBioscience, San Diego, CA, USA) [4]. The proportion of CD25highFoxp3? cells gated in the CD4? cell fraction was recorded as Foxp3? Tregs. The cells were analyzed on a FACSCaliburÒ flow cytometer (BD Biosciences) using CellQuest software.
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Transforming growth factor (TGF)-b measurement The TGF-b concentration in platelet-poor plasma was measured using the Luminex assay (Life Technology, Grand Island, NY, USA) according to the manufacturer’s instructions. Statistical analyses Continuous variables are shown as the mean ± standard deviation. Comparisons between two groups were tested for statistical significance using the nonparametric Mann– Whitney U test.
Results and discussion Treg-deficient ITP mice were treated with rTPO or vehicle and followed until week 10 (Fig. 1a). Platelet counts gradually increased after rTPO administration and peaked at week 7. After this time point, platelet counts started to decrease, but increased again at week 9 and remained high at week 10. Long-term observation of two rTPO-treated mice revealed that platelet recovery persisted for more than 20 weeks. Previous studies showed that increased platelet counts in rTPO-treated normal mice were no longer detectable 2 weeks after treatment [12] and that spontaneous platelet recovery was not observed in the ITP mouse model [4]. Thus, our finding, combined with those of previous reports, suggested that rTPO may exert plateletincreasing effects other than the direct stimulation of platelet production. To examine rTPO’s effects on immune regulation, we prepared splenocytes from rTPO- or mocktreated ITP mice at week 10 and evaluated their autoantibody production (Fig. 1b). While splenocytes from mocktreated mice spontaneously produced pathogenic IgG antiplatelet antibodies, those from rTPO-treated mice did not. This is consistent with a previous report showing that short-term treatment of male (NZW 9 BXSB) F1 mice, another ITP model, with rTPO resulted in reduction of platelet-associated IgG [13]. Next, we evaluated the autoreactive T-cell responses to a series of recombinant GPIba fragments (Fig. 1c). Splenic T cells from mock-treated mice proliferated in response to GPIba fragments, whereas those from rTPO-treated ITP mice showed no detectable response, but did respond to the mitogenic stimulation with PHA. Together, these findings indicate that short-term treatment of ITP mice with thrombopoietic agents not only stimulates platelet production, but also induces immune tolerance to platelet autoantigens. In our mouse model, transferred conventional CD4? T cells, which were confirmed to lack Foxp3? Tregs, expand rapidly through homeostatic proliferation, while
TPO induces immune tolerance in ITP mice
Fig. 1 Short-term treatment with rTPO in a Treg-deficient mouse ITP model. a Serial platelet counts in rTPO- or vehicle (mock)-treated ITP mice. The broken line indicates the cutoff level for thrombocytopenia, defined as a platelet count of 0.33 9 106/lL, and the arrowheads denote the timing of administration of rTPO or vehicle. Asterisks denote statistically significant differences between rTPOand mock-treated mice. b IgG anti-platelet antibodies in the supernatants of splenocyte cultures derived from rTPO- and mocktreated mice at week 10. Antibody levels were determined by flow cytometry and are shown as the mean fluorescent index (MFI) ratio. c T-cell proliferative response to recombinant GPIba fragments in splenocyte cultures derived from rTPO- and mock-treated mice at week 10. Phytohemagglutinin (PHA) was used to demonstrate nonspecific T-cell responsiveness
the transfer of Treg-deficient CD4? cells results in the expansion of autoreactive T cells leading to a harmful anti-platelet autoimmune response [4]. In addition, adaptive Foxp3? Tregs can be peripherally induced from naive CD4? T cells in the presence of IL-2 and TGF-b [3]. To examine rTPO’s effect on this process in ITP mice, Foxp3? Tregs were evaluated using splenocytes derived from rTPO- and mock-treated ITP mice at week 10. Foxp3? Tregs were detected in both rTPO- and mocktreated mice, but their proportion was significantly greater in the rTPO-treated mice (Fig. 2), suggesting that shortterm rTPO treatment promotes the peripheral induction of Foxp3? Tregs. We also measured the circulating TGF-b before and 2 weeks after rTPO or mock treatment. TGF-b levels were significantly increased after rTPO treatment (0.45 ± 0.09 vs 3.03 ± 4.69 pg/mL, P \ 0.05), but not after mock treatment (0.58 ± 0.21 vs 0.41 ± 0.01 pg/
343
Fig. 2 Proportion of Foxp3? Tregs in splenocytes derived from rTPO- and mock-treated ITP mice at week 10. a A representative dot plot analysis of CD25highFoxp3? cells gated in the CD4? cell fraction. The upper-right quadrant corresponds to Foxp3? Tregs. b Proportion of Foxp3? Tregs in rTPO- and mock-treated ITP mice
mL). This effect is probably due to increased platelet and megakaryocyte counts, since treating ITP patients with eltrombopag results in increased circulating TGF-b levels in correlation with increased platelet numbers [14]. TGF-b is essential for maintenance of Treg function, and treatment with eltrombopag also improves Treg function in ITP patients [14]. Taken together, TGF-b released from platelets and megakaryocytes, which are expanded by treatment with thrombopoietic agents, may contribute to the differentiation of acquired Foxp3? Tregs and enhancement of their immunosuppressive functions. However, other mechanisms leading to immune tolerance such as exposure to high doses of antigen, an approach used to induce immune tolerance to exogenous factor VIII in hemophilia patients [15], also definitely play a role, since adaptive transfer of Tregs alone after onset of thrombocytopenia failed to increase platelet count in our mouse model [4]. In summary, short-term treatment with a thrombopoietic agent induced immune tolerance to platelet autoantigens in a mouse ITP model, suggesting that TPO-RAs may function in part by suppressing the immunopathogenic process of ITP. However, in ITP patients, sustained platelet recovery after discontinuation of TPO-RAs is much less common than in the animal model. Future studies will be required to identify factors controlling the immune tolerance induction in response to TPO-RAs in ITP patients.
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Acknowledgments This work was supported by a research grant on intractable diseases from the Japanese Ministry of Health, Labor, and Welfare. Conflict of interests
8.
The authors declare no conflict of interest. 9.
References 1. Semple JW, Provan D. The immunopathogenesis of immune thrombocytopenia: T cells still take center-stage. Curr Opin Hematol. 2012;19:357–62. 2. Kuwana M, Kaburaki J, Ikeda Y. Autoreactive cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura: role in production of anti-platelet autoantibody. J Clin Invest. 1998;02:1393–402. 3. Nishimoto T, Kuwana M. CD4?CD25?Foxp3? regulatory T cells in pathophysiology of immune thrombocytopenia. Semin Hematol. 2013;50:S43–9. 4. Nishimoto T, Satoh T, Takeuchi T, Ikeda Y, Kuwana M. Critical role of CD4(?)CD25(?) regulatory T cells in preventing murine autoantibody-mediated thrombocytopenia. Exp Hematol. 2012;40:279–89. 5. Nishimoto T, Satoh T, Simpson EK, Ni H, Kuwana M. Predominant autoantibody response to GPIb/IX in a regulatory T-cell-deficient mouse model for immune thrombocytopenia. J Thromb Haemost. 2013;11:369–72. 6. Kuter DJ. The biology of thrombopoietin and thrombopoietin receptor agonists. Int J Hematol. 2013;98:10–23. 7. Khellaf M, Michel M, Quittet P, et al. Romiplostim safety and efficacy for immune thrombocytopenia in clinical practice: 2-year
123
10.
11.
12. 13.
14.
15.
results of 72 adults in a romiplostim compassionate-use program. Blood. 2011;118:4338–45. Bussel JB, Buchanan GR, Nugent DJ, et al. A randomized, double-blind study of romiplostim to determine its safety and efficacy in children with immune thrombocytopenia. Blood. 2011;118:28–36. Ghadaki B, Nazi I, Kelton JG, Arnold DM. Sustained remissions of immune thrombocytopenia associated with the use of thrombopoietin receptor agonists. Transfusion. 2013;53:2807–12. Go´mez-Almague D, Herrera-Rojas MA, Jaime-Pe´rez JC, et al. Eltrombopag and high-dose dexamethasone as frontline treatment of newly diagnosed immune thrombocytopenia in adults. Blood. 2014;123:3906–8. Hattori N, Kuwana M, Kaburaki J, Mimori T, Ikeda Y, Kawakami Y. T cells that are autoreactive to b2-glycoprotein I in patients with antiphospholipid syndrome and healthy individuals. Arthritis Rheum. 2000;43:65–75. Miyazaki H. Physiologic role of TPO in thrombopoiesis. Stem Cells. 1996;14:133–8. Shibuya K, Kuwaki T, Tahara E, et al. Marked improvement of thrombocytopenia in a murine model of idiopathic thrombocytopenic purpura by pegylated recombinant human megakaryocyte growth and development factor. Exp Hematol. 2002;30:1185–92. Bao W, Bussel JB, Heck S, et al. Improved regulatory T-cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents. Blood. 2010;116:4639–45. Reipert BM, van Helden PM, Schwarz HP, Hausl C. Mechanisms of action of immune tolerance induction against factor VIII in patients with congenital haemophilia A and factor VIII inhibitors. Br J Haematol. 2007;136:12–25.
RAPID COMMUNICATION
Induction of immune tolerance to platelet antigen by short-term thrombopoietin treatment in a mouse model of immune thrombocytopenia Tetsuya Nishimoto • Miku Numajiri • Hisataka Nakazaki • Yuka Okazaki • Masataka Kuwana
Received: 26 June 2014 / Revised: 31 August 2014 / Accepted: 1 September 2014 / Published online: 12 September 2014 Ó The Japanese Society of Hematology 2014
Abstract Immune thrombocytopenia (ITP) is an autoimmune disorder caused by IgG anti-platelet autoantibodies. Thrombopoietin (TPO) receptor agonists are highly effective in inducing the recovery of platelet counts in ITP patients. Although these agents are thought to promote platelet production without affecting the autoimmune pathogenesis of the disease, a small subset of ITP patients exhibits sustained platelet recovery after treatment termination. To investigate mechanisms involved in this sustained recovery, we evaluated the effects of short-term TPO treatment using a mouse ITP model generated by Foxp3? T regulatory cell (Treg) depletion. After treatment, platelet recovery was sustained, along with complete suppression of both anti-platelet autoantibody production and T-cell responses to platelet autoantigens. TPO treatment also promoted the peripheral induction of Foxp3? Tregs in conjunction with elevated circulating TGF-b levels. In summary, thrombopoietic agents are capable of inducing immune tolerance to platelet autoantigens, thereby suppressing the autoimmune pathogenesis of ITP. Keywords Immune thrombocytopenia Immune tolerance Platelets T regulatory cells Thrombopoietin
T. Nishimoto M. Numajiri H. Nakazaki Y. Okazaki M. Kuwana Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Y. Okazaki M. Kuwana (&) Department of Allergy and Rheumatology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan e-mail: [email protected]
Introduction Immune thrombocytopenia (ITP) is an autoimmune disorder caused by the production of IgG autoantibodies to platelet membrane glycoproteins, such as GPIIb/IIIa and GPIb [1], which depends on activation of pathogenic autoreactive CD4? T cells [2]. In ITP patients, various immunosuppressive processes, including T regulatory cell (Treg)-mediated immune regulation, are dysfunctional, resulting in autoreactive T-cell activation [3]. We recently demonstrated that Foxp3? Treg-deficient mice spontaneously develop sustained thrombocytopenia associated with IgG anti-GPIb antibody production [4, 5], analogous to the pathophysiology of human ITP. This mouse model is useful for evaluating novel therapeutic strategies for ITP. Thrombopoietin (TPO) regulates thrombopoiesis through the activation of megakaryocytes in the bone marrow, resulting in increased platelet production [6]. Recently, the development of TPO receptor agonists (TPORAs), such as romiplostim and eltrombopag, was a significant breakthrough in ITP treatment [6]. It is thought that TPO-RAs do not affect the autoimmune pathogenesis of ITP, since platelet counts typically drop to pre-treatment levels immediately after treatment termination. However, recent reports showed that platelet recovery was unexpectedly sustained in some patients even after TPO-RA was discontinued [7–9]. The exact prevalence of TPO-RAinduced sustained remission is unclear, but one report described that this occurred in 3 of 31 patients treated with TPO-RAs [9]. Based on this observation, a single course of dexamethasone in combination with eltrombopag has been evaluated for a potential therapeutic strategy that induces long-term remission in newly diagnosed ITP patients [10]. Here, we investigated mechanisms mediating the sustained
123
342
effects of thrombopoietic agents using our mouse ITP model.
Materials and methods Recombinant TPO (rTPO) treatment of Treg-deficient ITP mice Treg-deficient mice were established by transferring CD4?CD25- T cells into syngeneic nude mice, as previously described [4]. Four weeks after transfer, ITP mice, which were confirmed to have thrombocytopenia (platelet count B0.33 9 106/lL), were treated with intravenous administration of mouse rTPO (300 ng/mouse; kindly provided by Kyowa Hakko Kirin, Tokyo, Japan) or vehicle for five consecutive days. Experimental protocols were approved by the Keio University Ethics Committee for Animal Experiments. Evaluation of autoantibody production and autoreactive T-cell responses in splenocytes The production of pathogenic IgG anti-platelet antibodies in splenocyte cultures was assessed using a platelet/IgGbinding assay, as described previously [4]. The T-cell response to GPIba was measured as previously described [2], with some modifications. Briefly, five different overlapping regions encompassing all 734 amino acid residues (aar) of GPIba were expressed and purified as recombinant maltose-binding protein (MBP) fusion proteins [11]. These included a1 (aar 18–250), a2 (aar 242–400), a3 (aar 399–570), a4 (aar 558–674), and a5 (aar 666–734). The splenocytes were cultured in triplicate with the recombinant GPIba fragments or MBP (5 lg/mL) for 7 days. T-cell proliferation was determined by 3H-thymidine incorporation, and the antigen-specific T-cell response was expressed as a stimulation index (SI), calculated as the ratio of the cpm incorporated in the presence of each GPIba fragment to the cpm incorporated with MBP. Phytohemagglutinin (PHA) was used to demonstrate non-specific T-cell responsiveness. Detection of Foxp3? Tregs Splenocytes were fixed, permeabilized, and incubated with fluorescence-conjugated monoclonal antibodies to CD4 (BD Biosciences, San Diego, CA, USA), CD25 (BD Biosciences), and Foxp3 (eBioscience, San Diego, CA, USA) [4]. The proportion of CD25highFoxp3? cells gated in the CD4? cell fraction was recorded as Foxp3? Tregs. The cells were analyzed on a FACSCaliburÒ flow cytometer (BD Biosciences) using CellQuest software.
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Transforming growth factor (TGF)-b measurement The TGF-b concentration in platelet-poor plasma was measured using the Luminex assay (Life Technology, Grand Island, NY, USA) according to the manufacturer’s instructions. Statistical analyses Continuous variables are shown as the mean ± standard deviation. Comparisons between two groups were tested for statistical significance using the nonparametric Mann– Whitney U test.
Results and discussion Treg-deficient ITP mice were treated with rTPO or vehicle and followed until week 10 (Fig. 1a). Platelet counts gradually increased after rTPO administration and peaked at week 7. After this time point, platelet counts started to decrease, but increased again at week 9 and remained high at week 10. Long-term observation of two rTPO-treated mice revealed that platelet recovery persisted for more than 20 weeks. Previous studies showed that increased platelet counts in rTPO-treated normal mice were no longer detectable 2 weeks after treatment [12] and that spontaneous platelet recovery was not observed in the ITP mouse model [4]. Thus, our finding, combined with those of previous reports, suggested that rTPO may exert plateletincreasing effects other than the direct stimulation of platelet production. To examine rTPO’s effects on immune regulation, we prepared splenocytes from rTPO- or mocktreated ITP mice at week 10 and evaluated their autoantibody production (Fig. 1b). While splenocytes from mocktreated mice spontaneously produced pathogenic IgG antiplatelet antibodies, those from rTPO-treated mice did not. This is consistent with a previous report showing that short-term treatment of male (NZW 9 BXSB) F1 mice, another ITP model, with rTPO resulted in reduction of platelet-associated IgG [13]. Next, we evaluated the autoreactive T-cell responses to a series of recombinant GPIba fragments (Fig. 1c). Splenic T cells from mock-treated mice proliferated in response to GPIba fragments, whereas those from rTPO-treated ITP mice showed no detectable response, but did respond to the mitogenic stimulation with PHA. Together, these findings indicate that short-term treatment of ITP mice with thrombopoietic agents not only stimulates platelet production, but also induces immune tolerance to platelet autoantigens. In our mouse model, transferred conventional CD4? T cells, which were confirmed to lack Foxp3? Tregs, expand rapidly through homeostatic proliferation, while
TPO induces immune tolerance in ITP mice
Fig. 1 Short-term treatment with rTPO in a Treg-deficient mouse ITP model. a Serial platelet counts in rTPO- or vehicle (mock)-treated ITP mice. The broken line indicates the cutoff level for thrombocytopenia, defined as a platelet count of 0.33 9 106/lL, and the arrowheads denote the timing of administration of rTPO or vehicle. Asterisks denote statistically significant differences between rTPOand mock-treated mice. b IgG anti-platelet antibodies in the supernatants of splenocyte cultures derived from rTPO- and mocktreated mice at week 10. Antibody levels were determined by flow cytometry and are shown as the mean fluorescent index (MFI) ratio. c T-cell proliferative response to recombinant GPIba fragments in splenocyte cultures derived from rTPO- and mock-treated mice at week 10. Phytohemagglutinin (PHA) was used to demonstrate nonspecific T-cell responsiveness
the transfer of Treg-deficient CD4? cells results in the expansion of autoreactive T cells leading to a harmful anti-platelet autoimmune response [4]. In addition, adaptive Foxp3? Tregs can be peripherally induced from naive CD4? T cells in the presence of IL-2 and TGF-b [3]. To examine rTPO’s effect on this process in ITP mice, Foxp3? Tregs were evaluated using splenocytes derived from rTPO- and mock-treated ITP mice at week 10. Foxp3? Tregs were detected in both rTPO- and mocktreated mice, but their proportion was significantly greater in the rTPO-treated mice (Fig. 2), suggesting that shortterm rTPO treatment promotes the peripheral induction of Foxp3? Tregs. We also measured the circulating TGF-b before and 2 weeks after rTPO or mock treatment. TGF-b levels were significantly increased after rTPO treatment (0.45 ± 0.09 vs 3.03 ± 4.69 pg/mL, P \ 0.05), but not after mock treatment (0.58 ± 0.21 vs 0.41 ± 0.01 pg/
343
Fig. 2 Proportion of Foxp3? Tregs in splenocytes derived from rTPO- and mock-treated ITP mice at week 10. a A representative dot plot analysis of CD25highFoxp3? cells gated in the CD4? cell fraction. The upper-right quadrant corresponds to Foxp3? Tregs. b Proportion of Foxp3? Tregs in rTPO- and mock-treated ITP mice
mL). This effect is probably due to increased platelet and megakaryocyte counts, since treating ITP patients with eltrombopag results in increased circulating TGF-b levels in correlation with increased platelet numbers [14]. TGF-b is essential for maintenance of Treg function, and treatment with eltrombopag also improves Treg function in ITP patients [14]. Taken together, TGF-b released from platelets and megakaryocytes, which are expanded by treatment with thrombopoietic agents, may contribute to the differentiation of acquired Foxp3? Tregs and enhancement of their immunosuppressive functions. However, other mechanisms leading to immune tolerance such as exposure to high doses of antigen, an approach used to induce immune tolerance to exogenous factor VIII in hemophilia patients [15], also definitely play a role, since adaptive transfer of Tregs alone after onset of thrombocytopenia failed to increase platelet count in our mouse model [4]. In summary, short-term treatment with a thrombopoietic agent induced immune tolerance to platelet autoantigens in a mouse ITP model, suggesting that TPO-RAs may function in part by suppressing the immunopathogenic process of ITP. However, in ITP patients, sustained platelet recovery after discontinuation of TPO-RAs is much less common than in the animal model. Future studies will be required to identify factors controlling the immune tolerance induction in response to TPO-RAs in ITP patients.
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Acknowledgments This work was supported by a research grant on intractable diseases from the Japanese Ministry of Health, Labor, and Welfare. Conflict of interests
8.
The authors declare no conflict of interest. 9.
References 1. Semple JW, Provan D. The immunopathogenesis of immune thrombocytopenia: T cells still take center-stage. Curr Opin Hematol. 2012;19:357–62. 2. Kuwana M, Kaburaki J, Ikeda Y. Autoreactive cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura: role in production of anti-platelet autoantibody. J Clin Invest. 1998;02:1393–402. 3. Nishimoto T, Kuwana M. CD4?CD25?Foxp3? regulatory T cells in pathophysiology of immune thrombocytopenia. Semin Hematol. 2013;50:S43–9. 4. Nishimoto T, Satoh T, Takeuchi T, Ikeda Y, Kuwana M. Critical role of CD4(?)CD25(?) regulatory T cells in preventing murine autoantibody-mediated thrombocytopenia. Exp Hematol. 2012;40:279–89. 5. Nishimoto T, Satoh T, Simpson EK, Ni H, Kuwana M. Predominant autoantibody response to GPIb/IX in a regulatory T-cell-deficient mouse model for immune thrombocytopenia. J Thromb Haemost. 2013;11:369–72. 6. Kuter DJ. The biology of thrombopoietin and thrombopoietin receptor agonists. Int J Hematol. 2013;98:10–23. 7. Khellaf M, Michel M, Quittet P, et al. Romiplostim safety and efficacy for immune thrombocytopenia in clinical practice: 2-year
123
10.
11.
12. 13.
14.
15.
results of 72 adults in a romiplostim compassionate-use program. Blood. 2011;118:4338–45. Bussel JB, Buchanan GR, Nugent DJ, et al. A randomized, double-blind study of romiplostim to determine its safety and efficacy in children with immune thrombocytopenia. Blood. 2011;118:28–36. Ghadaki B, Nazi I, Kelton JG, Arnold DM. Sustained remissions of immune thrombocytopenia associated with the use of thrombopoietin receptor agonists. Transfusion. 2013;53:2807–12. Go´mez-Almague D, Herrera-Rojas MA, Jaime-Pe´rez JC, et al. Eltrombopag and high-dose dexamethasone as frontline treatment of newly diagnosed immune thrombocytopenia in adults. Blood. 2014;123:3906–8. Hattori N, Kuwana M, Kaburaki J, Mimori T, Ikeda Y, Kawakami Y. T cells that are autoreactive to b2-glycoprotein I in patients with antiphospholipid syndrome and healthy individuals. Arthritis Rheum. 2000;43:65–75. Miyazaki H. Physiologic role of TPO in thrombopoiesis. Stem Cells. 1996;14:133–8. Shibuya K, Kuwaki T, Tahara E, et al. Marked improvement of thrombocytopenia in a murine model of idiopathic thrombocytopenic purpura by pegylated recombinant human megakaryocyte growth and development factor. Exp Hematol. 2002;30:1185–92. Bao W, Bussel JB, Heck S, et al. Improved regulatory T-cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents. Blood. 2010;116:4639–45. Reipert BM, van Helden PM, Schwarz HP, Hausl C. Mechanisms of action of immune tolerance induction against factor VIII in patients with congenital haemophilia A and factor VIII inhibitors. Br J Haematol. 2007;136:12–25.
